Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Fluorescent dyes are used to impart both visible color and fluorescence to other materials. The dyes of this invention are sulfonated derivatives of xanthene-based dyes that are typically fluorescein, rhodol or rhodamine derivatives.
"Fluorescein" dyes include derivatives of 3H-xanthen-6-ol-3-one that are typically substituted at the 9-position by a 2-carboxyphenyl group. "Rhodol" dyes include derivatives of 6-amino-3H-xanthen-3-one that are typically substituted at the 9-position by a 2-carboxyphenyl group. "Rhodamine" dyes include derivatives of 6-amino-3H-xanthen-3-imine that are typically substituted at the 9-position by a 2-carboxyphenyl group. ##STR1##
Rhodols, rhodamines and fluoresceins are typically substituted by a derivative capable of forming a 5- or 6-membered lactone or lactam ring. For example in the case of fluorescein the spirolactone form of the dye has the structure: ##STR2##
Sulfonation of some cyanine dyes has been shown to decrease the inherent tendency of those dyes to form dimers and aggregates, presumably due to the increased polar character imparted by the sulfonic acid moiety (Waggoner et al. U.S. Pat. No. 5,268,486 (1993)). Ring sulfonation increases the brightness of some fluorescent dyes, including carbocyanine dyes (co-pending application Ser. No. 08/702,396 by Leung et al., filed Aug. 14, 1996) and coumarin dyes (co-pending application Ser. No. 08/749,753 by Wang et al., filed Nov. 15, 1996, now allowed).
The present invention describes xanthene dyes, including fluorescein, rhodol and rhodamine dyes, that are substituted by at least one sulfonate moiety. The sulfonated xanthene dyes of the invention possess considerable advantages over their non-sulfonated analogs. In particular, their fluorescence yields are typically higher than those of other dyes having comparable spectra, including fluorescein, CY-2, tetramethylrhodamine, CY-3 and TEXAS RED dye (Table 5). The sulfonated dyes of the invention exhibit resistance to quenching upon protein conjugation (FIG. 3). In addition, the dyes of the invention possess better photostability than fluorescein or the sulfonated cyanine CY2 and CY3 dyes (as shown in FIG. 6). The spectra of the sulfonated rhodamine dyes of the invention are insensitive to pH changes in the range between pH 4 and 10. Also, the dyes of the invention possess substantially greater water solubility than non-sulfonated analogs.